Preparation method of aquaporin AqpZ

An aquaporin and fusion protein technology, applied in the field of aquaporin AqpZ, can solve the problems of difficult to meet the structural research and prepare a water recovery device, and achieve the effect of avoiding toxicity

Inactive Publication Date: 2013-06-26
ZHEJIANG UNIV
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  • Abstract
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Problems solved by technology

Therefore, it is difficult to meet the requirements of structural studies and fabrication of aquaporin-based water recycling devices

Method used

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  • Preparation method of aquaporin AqpZ
  • Preparation method of aquaporin AqpZ
  • Preparation method of aquaporin AqpZ

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Embodiment Construction

[0032] The preparation method of aquaporin AqpZ of the present invention comprises the following steps:

[0033] 1. Amplification of E. coli aquaporin AqpZ-HIS: Two pairs of primers were designed according to the E. coli aquaporin gene sequence on NCBI. Eight HIS affinity tags were artificially added to the 3' end of the AqpZ gene by designing primers. The oligonucleotide sequence of the upstream primer of the first primer is shown in SEQ ID No.1, the oligonucleotide sequence of the downstream primer of the first primer is shown in SEQ ID No.2, and the oligonucleotide sequence of the upstream primer of the second primer is shown in SEQ ID No.2. The nucleotide sequence is shown in SEQ ID No.3, and the oligonucleotide sequence of the downstream primer of the second primer is shown in SEQ ID No.4. According to the characteristics of the multiple cloning sites of the two vectors pMAL-p2 and pMAL-c5e, add the upstream and downstream of the first primer respectively Bam HI and ...

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Abstract

The invention discloses a preparation method of an aquaporin AqpZ. The method comprises the following steps: 1, amplifying Escherichia coli aquaporin AqpZ-HIS; 2, constructing a recombinant expression vector, carrying out construction and inducible expression of engineering strains, and carrying out ultrasonic fragmentation to obtain fusion protein MBP-AqpZ-HIS thalli; and 3, purifying to obtain the aquaporin AqpZ. The method realizes the efficient expression of a maltose-binding protein-aquaporin AqpZ-His-tag fusion protein in Escherichia coli, and overcomes problems comprising the toxicity of traditional AqpZ over-expression to host cells and insoluble inclusion body formation; and high-purity aquaporin AqpZ can be obtained only through two-time simple affinity chromatography separation of the aquaporin expressed through the method, so the method is simple to operate and is suitable for the industrialized production of the aquaporin AqpZ.

Description

technical field [0001] The present invention relates to a protein expression and purification method, more particularly, to a method for obtaining aquaporin AqpZ by expressing and purifying in Escherichia coli using a maltose binding protein / polyhistidine binary tag expression system. Background technique [0002] Transmembrane transport of water molecules is one of the fundamental activities of life. The rapid transmembrane transport of water molecules is an important factor for organisms to rapidly adapt to changes in the osmotic pressure of the extracellular environment. Aquaporins are one of the important members of important intrinsic proteins. It is a cell-specific transmembrane channel for water molecules to enter and leave cells, so it plays an important role in maintaining the homeostasis of the intracellular environment. Aquaporins are found throughout the biological kingdom, and water channels have been identified from prokaryotic bacteria to eukaryotic plants a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/70C07K14/245C07K1/22
Inventor 徐志南潘剑峰黄磊蔡谨
Owner ZHEJIANG UNIV
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